This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Brassinosteroids (BRs) are plant steroid hormones, which play important roles in a wide range of physiological and developmental processes, including cell expansion, cell division, vascular differentiation, seed germination, photomorphogenesis, stress tolerance and disease resistance. Molecular genetic studies have identified several BR signal transduction components of the BR signaling pathway, through which BRs bind the cell surface receptors to regulate nuclear gene expression. However, there are still gaps in the current BR signaling pathway. In particular, the substrates of the receptor kinases that transduce the BR signal from plasma membrane to cytoplasmic components remain unknown. Despite the success of genetic studies, many genes'function cannot be discovered by forward genetic approaches, largely due to genetic redundancy. Therefore, alternative approaches are required to significantly advance our understanding of the BR signaling pathway. Thus, we have performed proteomic studies and identified BR-regulated proteins in Arabidopsis using two-dimensional difference gel electrophoresis (2-D DIGE) followed by mass spectrometry. This study aims at characterizing plasma membrane-localized BR-responsive proteins, including the BR-signaling kinases (BSKs). Functional studies of these proteins have demonstrated their key role as BRI1 substrate in the BR signaling pathway. We plan to identify additional interacting proteins of BSKs using affinity purification followed by mass spectrometry analysis. Such analysis will advance our understanding of BR signal transduction and provide molecular tools for improving plant productivity.